The present invention relates to inhibition of inflammation by administration of a pharmacologically active ureido derivative of poly-4-amino-2-carboxy-1-methyl pyrrole.
Inflammatory reactions are serious medical indications arising from a variety of conditions. Essential to the early inflammatory response is the selective recruitment of leukocytes into the affected tissue(s). This process is controlled, in part, by chemokines, which are small (8-10 kD), inducible cytokines that act primarily as chemoattractants and activators of specific types of leukocytes in a variety of immune and inflammatory responses (Oppenheim et al., Ann. Rev. Immunol., 9, 617-648 (1991); and Taub et al., Cytokine, 5, 175-179 (1993)). Chemokines are produced in response to an array of factors, including viruses, bacterial products, IL-1, TNF, C5a, LTB4, and IFNs (Strieter et al., J. Immunol. 156, 3583-3586 (1996)). Chemokines have been detected during inflammation in the skin, brain, joints, meninges, lungs, blood vessels, kidneys, and gastrointestinal tract. Within these organs, chemokines have been identified in many types of cells, indicating that most cells secrete chemokines given the appropriate stimulus.
Chemokines have been subdivided into families based on the arrangement of the conserved cysteine residues of the mature proteins (Baggiolini et al., Adv. Immunol., 55, 97-179 (1994); and Baggiolini et al., Ann. Rev. Immunol., 15, 675-705 (1997). The xcex1- and xcex2-chemokines, which contain four conserved cysteines, belong to the largest families. The CXC or xcex1-chemokines are those which have one amino acid residue separating the first two conserved cysteine residues, whereas the CC or xcex2-chemokines are those in which the first two conserved cysteine residues are adjacent. Additionally, there are at least two other families, including the C or xcex3-chemokines, which lack two (the first and third) of the four cysteine residues (Kelner et al., Science, 266, 1395-1359 (1994)), and the CXXXC chemokines, in which the first two cysteine residues are separated by three amino acids (Bazan et al. Nature, 385, 640-644 (1997)).
Within the CXC or xcex1-chemokine family, there are chemokines that contain a characteristic glutamic acid-leucine-arginine (ELR) sequence immediately preceding the first cysteine residue near the N terminus and those that lack this sequence (Clark-Lewis et al., J. Biol. Chem., 266, 23128-23134 (1991)). CXC chemokines possessing the ELR sequence (e.g., human IL-8, mouse KC, mouse MIP-2, mouse LIV, ENA-78, GCP-2, and GROxcex1, xcex2 and xcex3) are chemoattractants and activators of neutrophils, whereas CXC chemokines lacking the sequence (e.g., IP10/mouse CRG, PBSF/SDF-1, and PF4) act primarily on lymphocyte populations. The CC or xcex2-chemokines (e.g., MIP-1xcex1, MIP-1xcex2, HCC-1, LEC, TARC, Eotaxin and RANTES) and the C or xcex3-chemokines (e.g., Lymphotactin) chemoattract and activate monocytes, lymphocytes, dendritic cells, eosinophils, and basophils with variable selectivity.
Chemokines mediate their chemotactic and other activities by binding to specific G-protein-coupled cell-surface receptors on target cells (Premack et al., Nat. Med., 2, 1174-1178 (1996); and Murphy et al., Ann. Rev. Immunol, 12, 593-633 (1994)). Like other G-protein-coupled receptors, chemokine receptors are functionally linked to phospholipases through G proteins and receptor activation leads to, among other things, the generation of inositol triphosphate, the release of intracellular calcium, and the activation of protein kinase C (Lodi et al., Science, 263, 1762-1767 (1994)). To date, five human CXC chemokine receptors (CXCR1 through CXCR5), eight human CC chemokine receptors (CCR1 through CCR8), and one human CXXXC chemokine receptor (CX3CR1) have been identified. While some receptors are restricted to certain cell types, others are widely expressed on a variety of cells. Further, chemokine receptors may be constitutively expressed on some cells and inducible on others, and may also be sensitive to the state of cell activation and differentiation. Finally, some chemokine receptors are also expressed on nonhematopoietic cells, including neurons, astrocytes, epithelial cells, and endothelial cells, suggesting further roles for the chemokine system.
Additionally, recent work has shown that some viral genomes are capable of encoding chemokine and chemokine receptor homologues (Gao et al., J. Biol. Chem., 269, 28539-28542 (1994); and Ahuja et al., J. Biol. Chem., 268, 20691-20694 (1993)). For example, the open reading frame (ORF) US28 of the human Cytomegalovirus (CMV) encodes a protein that shares approximately 30% sequence homology with the CC chemokine receptor CCR-1 and is capable of binding the MIP-1xcex1, MIP-1xcex2, MCP-1 and RANTES chemokines in vitro. Similarly, the ORF ECRF3 of Herpes saimiri encodes a protein that is 30% homologous with the IL-8 chemokine receptors and able to bind IL-8, GRO-xcex1, GRO-xcex2 and NAP-2 chemokines. Other human herpesviruses (HHV) also have been shown to express chemokine-receptor homologues that can bind human chemokines (Luster, New Engl. J. Med., 338, 436-445 (1998); Soldan et al., Nature Med., 3, 1394-1397 (1997); and Wells et al., TIPS, 19, 376-379 (1998)). For example, Kaposi""s sarcoma-associated HHV8 expresses receptor homologues that cause the infected cell to respond to CXC chemokines, such as IL-8, SDF-1xcex1 and IP10. The HHV6 virus, which has been found in nervous system tissue characterized by the active myelin destruction associated with multiple sclerosis (Soldan (1997), supra), encodes a xcex2-chemokine receptor that is capable of binding MIP-1xcex1, MIP-1xcex2, RANTES, and MCP-1 chemokines (Isegawa et al., J. Vir., 72, 6104-6112 (1998)). Significantly, in contrast to other viruses, such as the HIV-1 virus, which infects its host""s cells via the host""s chemokine receptors, the herpesviruses enter their host""s cells via the Pol receptor. Once the herpesvirus gains entry into the cell, it produces virally encoded chemokines and chemokine receptors, which are effectively masked from the host cell""s immune system.
The secretion of chemokines has been detected in a wide variety of diseases characterized by inflammatory reactions resulting from the selective accumulation and activation of leukocytes in the affected tissue(s) (see Strieter (1996), supra). The type of inflammatory infiltrate that characterizes a specific disease is controlled, in part, by the type of chemokines expressed in the diseased tissue. For example, patients with acute respiratory distress syndrome, which is characterized by a massive influx of neutrophils into the tissue, exhibit an elevated concentration of potent neutrophil chemoattractants. Recently, it has been shown that IL-8 production is also increased in reperfusion injury, which similarly involves the recruitment and activation of neutrophils (Strieter (1996), supra; and Karakurum et al., J. Clin. Invest., 93, 1564-1570 (1994)). Patients suffering from asthma demonstrate a selective accumulation and activation of eosinophils in lung tissue, correlating with an elevated level of Eotaxin, RANTES, and MIP-1xcex1. Similarly, monocyte chemoattractant proteins play an important role in allergic inflammation, which is also characterized by the activation and migration of eosinophils into the affected tissue(s). Other disease states associated with inflammatory responses mediated through chemokines include arthritis, non-bacteria-mediated respiratory distress syndrome, and blunt force trauma, as well as the demyelination of nerve cells associated with multiple sclerosis.
Those skilled in the art will appreciate that prior art methods of alleviating or mitigating the negative effects of inflammation are limited. This presents a serious problem for those patients who can not tolerate current available medications, such as aspirin, which is used to treat blunt force trauma, or anti-inflammatory agents, which are used to treat allergies. Accordingly, there is a need for new methods of lessening inflammation, in particular non-TNF-dependent inflammation.
Ureido derivatives of substituted pyrroles, a class of compounds regarded as Distamycin A derivatives, are known to inhibit angiogenesis and HIV replication. For example, see U.S. Pat. No. 5,420,296 (Mongelli et al.) and Clanton et al., Antiviral Research, 27, 335-354 (1995). Additionally, it has been suggested that some of these compounds might be used to treat a disease state in which TNF-xcex1 plays a detrimental role in the pathology (see U.S. Pat. No. 5,260,329 (Mongelli et al.)). Surprisingly, it has now been discovered that the dimeric ureido derivatives of poly-4-amino-2-carboxy-1-methyl pyrrole are useful for inhibiting inflammation, in particular non-TNF-xcex1 dependent inflammation.
In view of this, it is an object of the present invention to provide a method of inhibiting inflammation, in particular inflammation that is not mediated by TNF-xcex1. This and other objects and advantages of the present invention will become apparent front the description provided herein.
The present invention provides a method of inhibiting inflammation, particularly non-TNF-xcex1 dependent inflammation, by administering an inflammation-inhibiting effective amount of a ureido derivative of a substituted pyrrole of the formula (I): 
wherein m and n are the same and each is an integer of 1 to 6; W is oxygen or sulphur; each of the B groups, which need not be, but preferably are the same, is (a) a saturated or an unsaturated carbocyclic ring system substituted by one or more acid groups (b) a saturated or an unsaturated, heteromonocyclic or heteropolycyclic ring, containing one or more heteroatoms chosen from nitrogen, oxygen, and sulfur, substituted by one or more acid groups; (c) a pyranyl or furanyl sugar residue substituted by one or more acid groups; or (d) a xe2x80x94CH2(CHA)rCH2A group, wherein each A group, being the same or different, is an acid group and r is 0, 1 or 2; each occurrence of D is independently selected and is N or CH; or a pharmaceutically acceptable salt thereof. Upon administration of the compound or a pharmaceutically acceptable salt thereof, the inflammation is inhibited.
The present invention provides a method of inhibiting inflammation, in particular non-TNF-xcex1 dependent inflammation, by administering to a mammal in need thereof (or at risk of developing an indication advantageously treated by reduction of inflammation) an inflammation-inhibiting effective amount of a compound of the formula (I): 
wherein m and n are the same and each is an integer of 1 to 6, and preferably an integer of 2 to 4; W is oxygen or sulphur; each occurrence of D is independently selected and is N or CH; each of the B groups, which need not be the same, but preferably are the same, is
(a) a saturated or an unsaturated carbocyclic ring system substituted by one or more acid groups;
(b) a saturated or an unsaturated, heteromonocyclic or heteropolycyclic ring, containing one or more heteroatoms chosen from nitrogen, oxygen, and sulfur, substituted by one or more acid groups;
(c) a pyranyl or furanyl sugar residue substituted by one or more acid groups; or
(d) a xe2x80x94CH2(CHA)rCH2A group, wherein each A group, being the same or different, is an acid group and r is 0, 1 or 2; or a pharmaceutically acceptable salt thereof, whereupon administration of the compound or a pharmaceutically acceptable salt thereof to the mammal, the inflammation is inhibited.
When two or more acid groups are present on a B group, as defined above under (a), (b) and (c), they can be the same or different. Examples of acid groups according to the definition of a B group given above under (a), (b), (c) and (d) for instance can be those chosen from the group consisting of sulfonic, sulfuric, sulfamic, sulfinic, phosphoric, phosphonic, phosphamic and carboxylic acid groups, i.e. SO3H, SO4H, SO3NH2, SO2H, PO4H2, PO3H2, PO2NH3 and CO2H. Preferably the are selected from the group consisting of sulfonic, sulfinic, phosphonic, phosphamic, and carboxylic acid groups.
Preferably, B is as defined above under (a). Preferably, when B is as defined above under (a), (b) and (c), it is substituted by 1 to 3 acid groups. When B is as defined above under (a) and (b), preferably it has 1 to 5 rings, and more preferably 1 to 3 rings. The rings defined under (a) and (b) preferably have 4 to 10 ring atoms, more preferably 5 to 8 ring atoms, and most preferably 5 or 6 ring atoms in each ring. Additionally, the rings of the ring system are preferably fused, as in naphthalene. Aromatic examples of B, defined above under (a), include phenyl, naphthyl, indenyl, anthracenyl, phenanthrenyl, benzonaphthyl, and fluorenyl. When B is a ring as defined above under (b) it is, for example, tetrahydropyranyl, tetrahydrofuranyl, furanyl, thiophenyl, pyrrol, oxazolyl, indenyl, benzofuranyl, benzopyronyl, quinolinyl, purinyl, or pyrimidinyl. Preferably, B is naphthyl. Preferably, the naphthyl is substituted with 1, 2 or 3 acid groups selected from sulfonic acid and phosphonic acid. When B is a sugar residue, as defined above under (c), it is, for example, a residue derived from glucosyl or ribosyl. When B is a group as defined above under (d), r is preferably 2.
Examples of pharmaceutically acceptable salts are either those with inorganic bases, such as sodium, potassium, calcium and aluminium hydroxides, or with organic bases, such as lysine, arginine, N-methyl-glucamine, triethylamine, triethanolamine, dibenzylamine, methylbenzylamine, di-(2-ethyl-hexyl)-amine, piperidine, N-ethylpiperidine, N,N-diethylaminoethylamine, N-ethylmorpholine, xcex2-phenethylamine, N-benzyl-xcex2-phenethylamine, N-benzyl-N,N-dimethylamine and the other acceptable organic amines.
Preferred compounds according to the present invention include the compounds of formula (I), wherein: each of m and n, being the same, is 2-4; W is oxygen; the B groups are the same and each is (axe2x80x2) an unsaturated carbocyclic ring system substituted by 1 to 3 acid groups; (bxe2x80x2) a tetrahydropyranyl or tetrahydrofuranyl ring substituted by 1 to 3 acid groups; or (cxe2x80x2) a glucosefuranosyl residue substituted by 1 to 3 acid groups; and the pharmaceutically acceptable salts thereof.
Some specific examples of preferred compounds useful in the context of the invention are the following: 
wherein B is: 
and the pharmaceutically acceptable salts thereof.
Other preferred compounds for use in the present inventive method include: 
These compounds are also known in the art by the NSC Nos. 645793, 645794, 651015, 651016, 651017, 658434, 662162, 668535, 668536, and 668537, respectively. Additionally, the value of m and n in these compounds is two. Other well-known compounds of formula I, wherein m and n are larger (e.g., 3) or smaller (e.g., 1), include 664740, 664739, 670886, 670887, and 670888 (see Clanton et al., supra).
The compounds of the present invention, and the salts thereof, can be prepared by a process comprising reacting a compound of formula (II): 
wherein n and B are as defined above with respect to formula (I), or a salt thereof, with a compound of formula (III): 
wherein W is as defined above with respect to formula (I), and each of the X groups, which can be the same or different, is a good leaving group. As is well known in the art, suitable salts of formula (I) can be obtained, as well as the corresponding free acid of formula (I). A salt of a compound of formula (II) can be a salt with inorganic bases, for example those mentioned above with respect to pharmaceutically acceptable salts. Sodium and potassium salts are among the preferred salts of the invention. Preferred examples of good leaving groups, denoted as X in formula (III), are halogen atoms, in particular chlorine, or other easily displaceable groups, such as imidazolyl, triazolyl, p-nitrophenoxy and trichlorophenoxy.
The reaction of a compound of formula (II), or a salt thereof, with a compound of formula (III) can be carried out according to well-known methods. When X is a halogen atom, the reaction is preferably carried out at a molar ratio of compound (II):compound (III) from about 1:1 to about 1:4. The reaction can be performed in organic solvents, such as dimethylsulfoxide, hexamethylphosphotriamide, dimethylacetamide or, preferably, dimethylformamide, or their aqueous mixtures, or in water/dioxane or water/toluene mixtures, in the presence of either an organic base (e.g., triethylamine or diisopropylethylamine), or an inorganic base (e.g., sodium bicarbonate or sodium acetate). The reaction temperature can vary from about xe2x88x9210xc2x0 C. to about 50xc2x0 C. with a reaction time from about 1 hr to about 12 hrs.
The compounds of formula (I) prepared according to the above-described procedures can be purified by any suitable technique. Such techniques include conventional methods, such as silica gel or alumina column chromatography, and/or by re-crystallization from organic solvents (e.g., lower aliphatic alcohols or dimethylformamide).
The compounds of formula (II) can be obtained according to known procedures. For instance, a compound of formula (II) can be obtained by reduction of a compound of formula (IV): 
wherein n and B are as defined above, by methods wellknown in the art. For example, the compounds of formula (IV) can be obtained by reacting an amine of formula Bxe2x80x94NH2, wherein B is defined as above with respect to formula (I), with a compound of formula (V): 
wherein n and X are as defined above with respect to formula (I).
The reaction of an amine of formula Bxe2x80x94NH2 with a compound of formula (V) is also a well-known process. Alternatively, a compound of formula (IV), wherein n is 2 or more, can be obtained by a multi-step-process comprising reacting a compound of formula (VI): 
wherein X is as defined above with respect to formula I, with an amine of formula Bxe2x80x94NH2, in which B is as defined above with respect to formula (I). The reaction, which can be carried out according to known methods, provides compounds of formula (VII): 
wherein B is as defined above with respect to formula (I).
A compound of formula (VII) is reduced according to known methods to provide a compound of formula (VIII): 
wherein B is as defined above with respect to formula (I), which in its turn is reacted with a compound of formula (VI), thus obtaining a compound of formula (IV), wherein n is 2. If a compound of formula (IV), wherein n is 3 or more is desired, a further reduction and acylation step is performed.
The compounds of formula (V) are known compounds and can be obtained, for example, according to Heterocycles, 27, 1945-1952 (1988).
The compounds of formula (VI) and the amine of formula Bxe2x80x94NH2 are known products and can be easily obtained according to known methods.
The compounds useful in the context of the present inventive method can be administered to an animal, especially a mammal (e.g., a mouse), and preferably a human, by any suitable means or routes. Such administration means or routes include, but are not limited to, intraperitoneally, subcutaneously, and intravenously. The compounds of the present invention should not ordinarily be administered orally, unless the inflammation occurs in the gastrointestinal tract, especially, before the stomach.
The dosage depends on the age, weight and condition of the patient and on the administration route. For example, a suitable dosage for systemic administration to adult humans in reasonably good health can range from about 0.5 to about 1000 mg per dose 1-4 times a day. For local administration (e.g., subcutaneously or topically), the compound should be present in a suitable concentration. For example, a solution of about 0.0001% to about 2.5% (weight/volume) of a compound of formula I can be injected at a site of local inflammation. Further, one skilled in the art will understand how to vary the dosage according to the particular needs of the patient based on the particular pharmacokinetics of the animal to be treated. For example, in the mouse, following intravenous administration of 50 mg/kg body weight, a peak concentration of about 700 xcexcM NSC 651016 is obtained. This concentration decreases with a half-life of about 50 hrs so that  greater than 10 xcexcM NSC 651016 remains in the circulation for over 24 hrs.
Pharmaceutical compositions useful in the context of the invention can comprise a compound of formula (I) as the active substance, in association with one or more pharmaceutically acceptable excipients and/or carriers. The pharmaceutical compositions of the invention are usually prepared following conventional methods and are administered in a pharmaceutically suitable form.
For instance, solutions for intravenous injection or infusion can contain as carrier, for example, sterile water or sterile aqueous isotonic saline solutions. Suspensions or solutions for intramuscular injections can contain, together with the active compound, a pharmaceutically acceptable carrier, e.g., sterile water, olive oil, ethyl oleate, glycols, e.g., propylene glycol, and, if desired, a suitable amount of lidocaine hydrochloride. In the forms for topical application, e.g., creams, lotions, or pastes for use in dermatological treatment, the active ingredient can be mixed with conventional oleaginous or emulsifying excipients.
While the method of the present invention is useful in the inhibition of inflammation in general, it is particularly useful in the inhibition of non-TNF-xcex1-dependent inflammation. Non-TNF-xcex1-dependent inflammation can be caused by any one of a number of conditions or disease states, such as asthma, allergy, blunt-force trauma, reperfusion injury, non-bacteria-mediated respiratory distress syndrome, and other conditions, such as the demyelination of nerve tissue, such as that which is associated with multiple sclerosis.
The method of inhibiting non-TNF-xcex1 dependent inflammationis particularly useful for inhibiting inflammation that is mediated through a chemokine. A review of the disease states associated with particular populations of chemokines, is found in Howard et al., Trends in Biotech., 14, 46-51 (1996). Furthermore, due to the known capacity of chemokines to activate selectively and control the movement of a specific population(s) of inflammatory cells, one with ordinary skill in the art can determine other inflammatory conditions that can be inhibited in accordance with the present inventive method by determining the population of inflammatory cells at the site of inflammation (e.g., by performing such assays as a blister test, an ELISA for fluid aspirates, or a biopsy for solid tumors). Alternatively, one with ordinary skill in the art can determine inflammatory conditions that can be inhibited in accordance with the present inventive method by determining which chemokines are present at the site of inflammation. Since many of the chemokine and chemokine receptor genes have been cloned and there are also monoclonal antibodies directed to many of the chemokines, one skilled in the art could perform Southern Blot, in situ hybridization, Western Blots or ELISA analysis to determine which chemokines are present in the inflamed tissue. Alternatively, the infiltrate in inflamed tissue can be examined, such as in accordance with the methods set forth in Examples 14 and 15.
The non-TNF-xcex1 dependent inflammation can be mediated through an xcex1-chemokine, such as SDF-1xcex1. Alternatively, the non-TNF-xcex1-dependent inflammation can be mediated through a xcex2-chemmokine, such as MIP-1xcex1, HCC-1, LEC, TARC, Eotaxin, or RANTES. The chemokine that mediates the non-TNF-xcex1-dependent inflammation can be encoded by a virus and can be determined in accordance with methods known in the art. If encoded by a virus, preferably the chemokine is encoded by a herpesvirus, such as CMV, Herpes saimiri, HHV8 or HHV6.
Also, in the method of inhibiting non-TNF-xcex1-dependent inflammation, the inflammation can be mediated through a chemokine receptor. The chemokine receptor can be an xcex1-chemokine receptor, such as CXCR4. The chemokine receptor alternatively can be a xcex2-chemmokine receptor, such as CCR1, CCR3, CCR5, and CCR8. Further, the xcex1- or xcex2-chemokine receptor can be encoded by a virus. The virally encoded xcex1- or xcex2chemokine receptor is preferably encoded by a herpesvirus, such as CMV, Herpes saimiri, HHV8 or HHV6.